Case Study 2. Practical Analytical Considerations for Conducting In Vitro Enzyme Kinetic Studies

The objective of this case study is to answer the following questions: (a) Why is it necessary to determine lower and upper limits of quantification (LLOQ and ULOQ, respectively) of a bioanalytical assay specifically for enzyme kinetic assays? How do you utilize LLOQ and ULOQ to correctly interpret your kinetic data? (b) Why should one use a linear fit and not a quadratic fit for standard curves? (c) Is quantification of an analyte possible without a reference standard? Can one assume equal signal intensities regardless of analytical technique (MS, UV)? (d) In the absence of reference standards, can you still determine kinetic constants? (e) With the need to keep substrate depletion at less than 20 % for linearity assumptions, does bioanalytical variability matter? (f) What buffer do you use for y...
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